The present invention relates to prosthetic grafts which are used to contain blood flow in vivo.
Diseases of the major circulatory and renal organs and vessels have created a need for prosthetic grafts to bypass, repair and/or replace the function of the diseased organs and vessels. Such grafts should ideally be non-immunogenic, non-calcific, and readily capable of recreating or reestablishing the natural blood contact interface of the organ or vessel to be replaced or repaired. Complications that have inhibited the widespread use of prosthetic grafts in organs and vessels in contact with blood include: (1) intimal hyperplasia, whereby smooth muscle cell and myofibroblast proliferation and extracellular matrix accumulation cause thickening of the intima in the graft and in the adjoining vessels, and ultimately lead to failure of the graft; and, (2) occlusion of the graft, whereby platelet adhesion and activation at the lumenal surface of the graft initiates thrombosis which, particularly in smaller bore vessel grafts, typically leads to complete occlusion of the graft.
Research over the past several decades has yet to produce a synthetic or biosynthetic small bore vascular graft which can approach the patency rates of autologous vessels. Since small bore grafts have a higher surface area to volume ratio and lower flow rates than larger grafts, the interaction of the graft with the blood is much greater. Platelet adhesion and activation at the lumenal surface of the graft are much more likely to result in complete graft occlusion. Larger vascular grafts are able to remain patent despite a layer of clot lining the lumen because this layer of clot undergoes constant remodeling and essentially maintains a constant thickness. In contrast, clotting on the surface of a graft smaller than 6 mm in inner diameter has a snowball effect and results in a continuous growth of the surface clot until the entire graft is occluded.
Currently, non-synthetic or biological small bore grafts are routinely used as an arterial replacement since nothing has proven to perform nearly as well as the autologous saphenous vein or internal mammary artery, which are the conventional biological materials used as a small diameter vascular graft. The use of these vessels requires additional surgery, particularly in the case of the saphenous vein, whereby the entire length of the leg must be opened to remove the vessel. The harvesting surgery increases the total operating time and can also lead to complications and discomfort. Furthermore, a small percentage of patients do not have autologous vessels suitable for harvesting. In some cases, the vessels are not available due to previous surgery, while in other cases, the vessel may be too small or varicose.
Even larger bore vessel and organ prosthetic grafts, however, suffer from complications associated with smooth muscle proliferation, compliance mismatch with native vessels, and poor endothelialization due to blood shear stresses and mechanical damage. Therefore, researchers have focused much effort on the development of bioinert and hemocompatible graft materials. However, a completely non-fouling surface has yet to be discovered and many now view the quest for such a material as unrealistic.
Rather than creating a non-fouling surface, others have focused on recreating the natural blood contacting interface in the body by seeding vascular grafts with endothelial cells (See for example, U.S. Pat. No. 5,723,324 to Bowlin et al.; U.S. Pat. No. 5,674,722 to Mulligan et al., U.S. Pat. No. 5,785,965 to Pratt et al., U.S. Pat. No. 5,766,584 to Edelman et al.). Although a small number of grafts seeded lumenally with endothelial cells have been implanted clinically outside of the United States, and improved patencies over non-seeded grafts have been observed, this approach has generally enjoyed mixed success, and the concept still faces many challenges. First, it is necessary that the cells used to seed the graft be autologous or otherwise non-immunogenic to avoid recognition and destruction of the cells by the patient""s immune system. To obtain autologous endothelial cells from a patient, the cells must be harvested from an isolated blood vessel. The harvesting surgical procedure not only increases prosthetic implant preparation time, but can also lead to complications and discomfort for the patient.
Second, retention of the cells on the graft surface after implantation has been an issue. A number of methods have been disclosed to address this issue, and include forcible injection of endothelial cells into the graft, preclotting and seeding the lumenal surface of the graft, static adhesion-seeding of the lumen, vacuum seeding of the lumen, seeding the lumen in an extracellular matrix, and seeding of the lumen using electrostatic and gravitational forces. These methods are reviewed or disclosed in more detail in U.S. Pat. No. 5,723,324, ibid. Additionally, it has been suggested that flow conditioning the seeded graft in vitro prior to implantation would improve cell retention by allowing the cells to secrete adhesion factors in response to slowly increasing shear rates (Dardik et al., 1999, J Vasc Surg 29: 157-67; Ballerman et al., 1995, Blood Purif 13: 125-34; and Ott and Ballerman, 1995, Surgery 117: 334-9). Although there is some evidence that methods such as conditioning may improve cell retention, all of these methods add yet another level of complexity to the seeding process and it is still not clear that significantly improved cellular retention can be achieved.
Therefore, there is a need for prosthetic grafts for use in the repair and replacement of vessels and organs in contact with blood flow that have improved long term patency and success rates, and which reduce the stress and discomfort experienced by the patient.
One embodiment of the present invention relates to a prosthetic graft for containment of blood flow in vivo. The graft includes: (a) a porous prosthetic implant for containing blood in vivo, the prosthetic implant having an outer surface that is not in contact with blood flow in vivo and an inner surface that is in contact with blood flow in vivo, the inner surface defining an interior space for containment of blood flow; and, (b) adherent cells adhered to the outer surface of the porous prosthetic implant. The adherent cells are transfected with at least one recombinant nucleic acid molecule operatively linked to a transcription control sequence, the recombinant nucleic acid molecule encoding a protein that enhances patency of said prosthetic implant.
The prosthetic implant can be configured as any blood containing vessel including, but not limited to, a prosthetic vessel, an artificial heart, a left ventricle assist device and/or a dialysis shunt. Prosthetic vessels include small, medium and large bore prosthetic vessels. Such vessels include venous and arterial prosthetic vessels. The implant can be constructed of any biological or non-biological material which includes, but is not limited to, highly resilient polyester, expanded polytetrafluorethylene (ePTFE), high porosity ePTFE, non-immunogenic xenogeneic tissue, porous silicon rubber, porous polyurethane, porous degradable polymer, and/or porous copolymers. In preferred embodiments, the prosthetic implant is non-immunogenic, non-calcific, and/or has a pore size of from about 0.1 xcexcm to about 500 xcexcm, and more preferably, from about 0.2 xcexcm to about 100 xcexcm.
The adherent cells can be any adherent cells and include, but are not limited to, fibroblasts, mesenchymal stem cells, bone marrow stem cells, embryonal stem cells, adipocytes, keratinocytes, vascular smooth muscle cells, platelets, and cells which have been genetically engineered to be adherent. In one embodiment, the cells are fibroblasts. The cells are transfected with at least one recombinant nucleic acid molecule encoding at least one protein that enhances patency of the prosthetic implant. Such proteins can include, but are not limited to, a protein that enhances angiogenesis in the vascular bed downstream of the prosthetic graft, a protein that enhances angiogenesis transmurally and into the interior space of the prosthetic implant to endothelialize the inner surface of the prosthetic implant, a protein that inhibits thrombosis, a protein that causes thrombolysis, a protein that inhibits smooth muscle migration and/or proliferation, and a vasodilator protein. Specific examples of such proteins are described in detail below.
In one embodiment of the present invention, the proteins are expressed by the adherent cells ex vivo, and secreted by the cells ex vivo and/or in vivo. In another embodiment, the proteins are expressed and secreted by the adherent cells in vivo. Preferably, the proteins are secreted into the pores of the implant and perfuse through the pores and into the inner surface of the implant. In one embodiment, the transcription control sequence includes an inducible promoter, so that the expression of the protein can be up- and/or down-regulated ex vivo or in vivo. Such an inducible promoter can be regulated, for example, by a compound that induces the promoter, including, but not limited to, an antibiotic, a hormone, a transcription factor and/or by a treatment such as internal or external radiation (e.g., X-ray).
Another embodiment of the present invention relates to a vascular graft, which includes: (a) a porous prosthetic vessel having a perivascular surface and a lumenal surface; and (b) adherent cells adhered to the perivascular surface of the porous prosthetic vessel. The adherent cells are transfected with at least one recombinant nucleic acid molecule operatively linked to a transcription control sequence, the recombinant nucleic acid molecule encoding a protein that enhances patency of the prosthetic vessel.
Yet another embodiment of the invention relates to a prosthetic graft for containment of blood flow in vivo which includes: (a) a porous prosthetic implant for containing blood in vivo, having an outer surface that is not in contact with blood flow in vivo and an inner surface that is in contact with blood flow in vivo, whereby the inner surface defines an interior space for containment of blood flow; and, (b) adherent cells adhered to the outer surface of the porous prosthetic implant. In this embodiment, the adherent cells express and secrete a protein that enhances patency of the prosthetic implant. In one aspect of this embodiment of the present invention, the adherent cells are endothelial cells that have been genetically modified to be adherent.
Yet another embodiment of the present invention relates to a method for producing a prosthetic graft, which includes the step of applying adherent cells to a porous prosthetic implant for containing blood in vivo, wherein the prosthetic implant has an outer surface and an inner surface that defines an interior space for containment of blood flow. The adherent cells are applied to the outer surface of the prosthetic implant. The adherent cells are transformed with at least one recombinant nucleic acid molecule operatively linked to a transcription control sequence, the recombinant nucleic acid molecule encoding a protein that enhances patency of the prosthetic implant. Other characteristics of the prosthetic implant are described above. Such a method preferably enhances naturally occurring endothelialization of the inner surface of the implant, inhibits thrombosis in the implant, inhibits thrombosis of the inner surface of the prosthetic implant due to smooth muscle migration and/or proliferation in the implant, and/or enhances formation of a neointima in the inner surface of the implant.
The step of applying can be performed by any method, including by a programmable mechanical graft rotator. When the implant is a prosthetic vessel, the step of applying includes seeding the outer surface of the vessel uniformly in both radial and longitudinal directions on the vessel. In one embodiment, the graft is incubated after the step of applying for about 5 minutes to about 14 days.
Another embodiment of the present invention relates to a method of implantation of a prosthetic graft for containment of blood flow. Such a method includes the step of implanting a prosthetic graft as described above into a patient. In one embodiment, the adherent cells are autologous to the patient. In another embodiment, the adherent cells are from a cell selected from the group of undifferentiated stem cell lines and/or embryonal cell lines.
If the transcription control sequence operatively linked to the at least one recombinant nucleic acid molecule includes an inducible promoter, the cells can be induced to express the protein either in vitro, prior to implantation of the graft into a patient, or in vivo, after implantation of the graft into a patient. Other characteristics of such a graft are described above.
Yet another embodiment of the present invention relates to a method for implantation of a prosthetic graft for containing blood flow in a patient. Such a method includes the steps of: (a) harvesting fibroblast cells from a patient in need of a prosthetic graft for containing blood flow; (b) transfecting the fibroblast cells with an isolated nucleic acid molecule encoding a protein that enhances patency of the graft; (c) applying the transfected fibroblast cells onto a surface of a prosthetic implant configured for containing blood flow in vivo for a time sufficient to allow the fibroblast cells to adhere to the surface to form a prosthetic graft, wherein the surface is not in contact with blood flow in vivo; and, (d) implanting the prosthetic graft into the patient.
Another embodiment of the present invention relates to a method of enhancing endothelialization of a vascular graft. Such a method includes the step of applying adherent cells to a porous prosthetic vessel having a perivascular surface and a lumenal surface, wherein the adherent cells are adhered to the perivascular surface of the prosthetic vessel. The adherent cells are transfected with at least one recombinant nucleic acid molecule operatively linked to a transcription control sequence, the recombinant nucleic acid molecule encoding a protein that enhances endothelialization of the prosthetic vessel. The protein is expressed and secreted by the adherent cells and perfuses through pores in the prosthetic vessel to the lumenal surface of the prosthetic vessel to enhance endothelialization of the graft at the inner surface.